Zeeuwen P L, Van Vlijmen-Willems I M, Jansen B J, Sotiropoulou G, Curfs J H, Meis J F, Janssen J J, Van Ruissen F, Schalkwijk J
Departments of Dermatology, Medical Microbiology, and Ophthalmology, University Medical Center St Radboud, Nijmegen, The Netherlands.
J Invest Dermatol. 2001 May;116(5):693-701. doi: 10.1046/j.1523-1747.2001.01309.x.
Using serial analysis of gene expression on cultured human keratinocytes we found high expression levels of genes putatively involved in host protection and defense, such as proteinase inhibitors and antimicrobial proteins. One of these expressed genes was the recently discovered cysteine proteinase inhibitor cystatin M/E that has not been characterized so far at the protein level with respect to tissue distribution and additional biologic properties. Here we report that cystatin M/E has a tissue-specific expression pattern in which high expression levels are restricted to the stratum granulosum of normal human skin, the stratum granulosum/spinosum of psoriatic skin, and the secretory coils of eccrine sweat glands. Low expression levels were found in the nasal cavity. The presence of cystatin M/E in skin and the lack of expression in a variety of other tissues was verified both at the protein level by immunohistochemistry or western blotting, and at the mRNA level by reverse transcriptase polymerase chain reaction or northern blotting. Using biotinylated hexapeptide probes we found that cystatin M/E is an efficient substrate for tissue type transglutaminase and for transglutaminases extracted from stratum corneum, and that it acts as an acyl acceptor but not as an acyl donor. Western blot analysis showed that recombinant cystatin M/E could be cross-linked to a variety of proteins extracted from stratum corneum. In vitro, we found that cystatin M/E expression in cultured keratinocytes is upregulated at the mRNA and protein level, upon induction of differentiation. We demonstrate that cystatin M/E, which has a putative signal peptide, is indeed a secreted protein and is found in vitro in culture supernatant and in vivo in human sweat by enzyme-linked immunosorbent assay or western blotting. Cystatin M/E showed moderate inhibition of cathepsin B but was not active against cathepsin C. We speculate that cystatin M/E is unlikely to be a physiologically relevant inhibitor of intracellular lysosomal cysteine proteinases but rather functions as an inhibitor of self and nonself cysteine proteinases that remain to be identified.
通过对培养的人角质形成细胞进行基因表达序列分析，我们发现了一些可能参与宿主保护和防御的基因的高表达水平，如蛋白酶抑制剂和抗菌蛋白。其中一个表达的基因是最近发现的半胱氨酸蛋白酶抑制剂胱抑素M/E，到目前为止，在蛋白质水平上，关于其组织分布和其他生物学特性尚未有相关描述。在此我们报告，胱抑素M/E具有组织特异性表达模式，其高表达水平仅限于正常人皮肤的颗粒层、银屑病皮肤的颗粒层/棘层以及小汗腺的分泌盘。在鼻腔中发现其表达水平较低。通过免疫组织化学或蛋白质印迹在蛋白质水平，以及通过逆转录聚合酶链反应或Northern印迹在mRNA水平，均证实了胱抑素M/E在皮肤中的存在以及在多种其他组织中的不表达。使用生物素化的六肽探针，我们发现胱抑素M/E是组织型转谷氨酰胺酶和从角质层提取的转谷氨酰胺酶的有效底物，并且它作为酰基受体而非酰基供体。蛋白质印迹分析表明，重组胱抑素M/E可以与从角质层提取的多种蛋白质交联。在体外，我们发现培养的角质形成细胞中胱抑素M/E的表达在诱导分化后在mRNA和蛋白质水平均上调。我们证明，具有假定信号肽的胱抑素M/E确实是一种分泌蛋白，通过酶联免疫吸附测定或蛋白质印迹在体外培养上清液中以及在体内人汗液中均能检测到。胱抑素M/E对组织蛋白酶B表现出中度抑制作用，但对组织蛋白酶C无活性。我们推测，胱抑素M/E不太可能是细胞内溶酶体半胱氨酸蛋白酶的生理相关抑制剂，而更可能是一种自我和非自我半胱氨酸蛋白酶的抑制剂，这些蛋白酶仍有待确定。
